Abstract The role of pyruvate kinase M2 isoform (PKM2) in tumor progression has been controversial. Previous studies showed that PKM2 promoted tumor growth in xenograft models; however, depletion of PKM2 in the Brca1-loss-driven mammary tumor mouse model accelerated tumor formation. Since oncogenic kinases are frequently activated in tumors and PKM2 phosphorylation promotes tumor growth, we hypothesized that PKM2 phosphorylation by activated kinases in tumor cells confers PKM2 oncogenic function, whereas non-phosphorylated PKM2 is non-oncogenic. Indeed, we found that PKM2 was phosphorylated at tyrosine 105 (Y105) in MDA-MB-231 breast cancer cells; whereas PKM2 was largely unphosphorylated in non-transformed MCF10A cells. Multiple kinases that are frequently activated in different cancer types were identified to phosphorylate PKM2-Y105 in our tyrosine kinase screening. Introducing the PKM2-Y105D phospho-mimetic mutant into MCF10A cells induced colony formation and the CD44hi/CD24neg cancer stem-like cell population partly by increasing YAP protein nuclear localization. ErbB2, a strong inducer of PKM2-Y105 phosphorylation, boosted YAP nuclear localization and cancer stem-like cell population. Lapatinib, an ErbB2 kinase inhibitor, decreased PKM2-Y105 phosphorylation and cancer stem-like cells, impeding PKM2 tumor-promoting function. Taken together, in non-transformed cells, unphosphorylated PKM2 is non-oncogenic; but in tumor cells, phosphorylated PKM2-Y105 by activated kinases gains oncogenic functions partly by activating YAP downstream signaling to increase cancer stem-like cell properties. Citation Format: Lin Zhang, Zhifen Zhou, Min Li, Dihua Yu. Oncogenic kinases-induced PKM2 tyrosine 105 phosphorylation converts nononcogenic PKM2 to a tumor promoter and induces cancer stem-like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2374.